Many environmental and societal benefits would result from the replacement of petroleum-based automotive fuels with renewable fuels obtained from plant materials (Lynd et al., (1991) Science 251:1318-1323; Olson et al., (1996) Enzyme Microb. Technol. 18:1-17; Wyman et al., (1995) Amer. Chem. Soc. Symp. 618:272-290). Each year, the United States bums over 120 billion gallons of automotive fuel, roughly equivalent to the total amount of imported petroleum. The development of ethanol as a renewable alternative fuel has the potential to eliminate United States dependence on imported oil, improve the environment, and provide new employment (Sheehan, (1994) ACS Symposium Series No. 566, ACS Press, pp 1-53).
In theory, the solution to the problem of imported oil for automotive fuel appears quite simple. Rather than using petroleum, a finite resource, ethanol, a renewable resource, can be produced efficiently by the fermentation of plant material. Indeed, Brazil has demonstrated the feasibility of producing ethanol and the use of ethanol as a primary automotive fuel for more than 20 years. Similarly, the United States produces over 1.2 billion gallons of fuel ethanol each year. Currently, fuel ethanol is produced from corn starch or cane syrup utilizing either Saccharomyces cerevisiae or Zymomonas mobilis (Z. mobilis). However, neither of these sugar sources can supply the volumes needed to realize a replacement of petroleum-based automotive fuels. In addition, both cane sugar and corn starch are relatively expensive starting materials, which have competing uses as food products.
Moreover, these sugar substrates represent only a fraction of the total carbohydrates in plants. Indeed, the majority of the carbohydrates in plants are in the form of lignocellulose, a complex structural polymer containing cellulose, hemicellulose, pectin, and lignin. Lignocellulose is found in, for example, the stems, leaves, hulls, husks, and cobs of plants. Hydrolysis of these polymers releases a mixture of neutral sugars including glucose, xylose, mannose, galactose, and arabinose. No known natural organism can rapidly and efficiently metabolize all of these sugars into ethanol.
Nonetheless, in an effort to exploit this substrate source, the Gulf Oil Company developed a method for the production of ethanol from cellulose using a yeast-based process termed simultaneous saccharification and fermentation (SSF) (Gauss et al. (1976) U.S. Pat. No. 3,990,944). Fungal cellulase preparations and yeasts were added to a slurry of the cellulosic substrate in a single vessel. Ethanol was produced concurrently during cellulose hydrolysis. However, Gulf's SSF process has some shortcomings. For example, the cell cycle time for yeast is relatively long (24-36 hours) and they are unable to ferment complex sugars. Further, fungal cellulases have to be added which have been considered, thus far, to be too expensive for use in large scale bioethanol processes (Himmel et al., (1997) Amer. Chem. Soc. pp. 2-45; Ingram et al., (1987) Appl. Environ. Microbiol. 53:2420-2425; Okamoto et al., (1994) Appl. Microbiol. Biotechnol. 42:563-568; Philippidis, G., (1994) Amer. Chem. Soc. pp. 188-217; Saito et al., (1990) J. Ferment. Bioeng. 69:282-286; Sheehan, J., (1994) Amer. Chem. Soc. pp 1-52; Su et al., (1993) Biotechnol. Lett. 15:979-984).
Moreover, producing ethanol using other organisms is difficult because pyruvate decarboxylase (PDC), a key enzyme for fermenting ethanol, is common only to plants, yeast, and fungi; and is rarely found in bacteria and is absent in animals (9, 25).